The general objectives of this proposal are to gain an education and basic laboratory training in molecular genetic methods and human genetics, in general, and to apply these skills to ophthalmologic disorders. The purpose is to contribute to the understanding and treatment of inherited ocular disorders, with a special emphasis on corneal diseases. These objectives will be met by attending graduate level courses and conferences as described (Appendix B-1) and by performing gene linkage studies in families with Reis Bucklers Corneal Dystrophy (RBCD). The RBCD proposal is the platform for my training to be an independent physician-scientist in the field of molecular genetics. RBCD is a dominantly inherited corneal disorder, has an infantile onset, complete penetrance and variable expressivity, and affects primarily central vision causing <20/200 visual acuity, disabling photophobia, and sharp stabbing pain from recurrent erosions in severely affected individuals. The slit lamp appearance of the milder cases closely resembles the reticular appearance of post EXCIMER laser corneal haze. A basic molecular understanding of RBCD may shed light on the more common problem of corneal scarring, the most common cause for corneal transplantation in the American elderly. Once the RBCD gene is cloned and characterized, the knowledge gained about the pathophysiologic mechanisms may lead to a greater understanding of corneal wound healing, scarring and recurrent erosion syndrome. Briefly, the following techniques are employed to perform gene-linkage analysis. First, the pedigree is ascertained and blood is collected from significant family members for DNA preparation. If needed, lymphoblast cell lines are established. Then, using highly informative microsatellite DNA markers, the pedigree is examined. Linkage analysis is performed in an effort to establish the chromosomal location of the gene for RBCD. Once linkage to a chromosome is found, fine structure linkage mapping, in conjunction with physical mapping, will be used to narrow down the region containing the RBCD locus. Yeast artificial chromosome (YAC) contigs which span this region will be constructed (or identified). A number of techniques such as exon trapping (Duyk 1990, Bucklers 1991), solution hybridization (Hoffman 1987),and detection of Hpall tiny fragment (HTF) islands (Conneally 1984) will then be used to identify expressed sequences from the RBCD region. Once the RBCD gene is cloned and characterized, understanding the pathophysiologic process of this anterior basement membrane disorder will be helpful in illuminating mechanisms in other corneal related conditions. In addition, this knowledge will assist in rational planning of diagnostic and counseling services as well as preventative therapies and treatment for affected patients.